Intra-Vacuolar Proliferation of F. Novicida within H. Vermiformis

Abstract

Francisella tularensis is a gram negative facultative intracellular bacterium that causes the zoonotic disease tularemia. Free-living amebae, such as Acanthamoeba and Hartmannella, are environmental hosts of several intracellular pathogens. Epidemiology of F. tularensis in various parts of the world is associated with water-borne transmission, which includes mosquitoes and amebae as the potential host reservoirs of the bacteria in water resources. In vitro studies showed intracellular replication of F. tularensis within A. castellanii cells. Whether ameba is a biological reservoir for Francisella in the environment is not known. We used Hartmannella vermiformis as an amebal model system to study the intracellular life of F. novicida. For the first time we show that F. novicida survives and replicates within H. vermiformis. The iglC mutant strain of F. novicida is defective for survival and replication not only within A. castellanii but also in H. vermiformis cells. In contrast to mammalian cells, where bacteria replicate in the cytosol, F. novicida resides and replicates within membrane-bound vacuoles within the trophozoites of H. vermiformis. In contrast to the transient residence of F. novicida within acidic vacuoles prior to escaping to the cytosol of mammalian cells, F. novicida does not reside transiently or permanently in an acidic compartment within H. vermiformis when examined 30 min after initiation of the infection. We conclude that F. tularensis does not replicate within acidified vacuoles and does not escape into the cytosol of H. vermiformis. The Francisella pathogenicity island locus iglC is essential for intra-vacuolar proliferation of F. novicida within H. vermiformis. Our data show a distinct intracellular lifestyle for F. novicida within H. vermiformis compared to mammalian cells.

Enhanced survival but not amplification of Francisella spp. in the presence of free-living amoebae

“…strain in the presence of FLA was not observed (Figures 1-3), which is contrary to the previous studies reporting increases in Francisella CFU densities following co-culture with FLA [33][34][35][36]. Specifically, 3−4 log 10 CFU mL −1 increases of LVS and Fn were observed by days 6−15 in the presence of Ac30010, Ac30234, and Vv at 25−30°C incubation and MOI of 10 [33,35,36]. However, in all of those studies, the assay buffer used was ATCC 712 medium, or peptone yeast extract (PYG) broth, which is the nutrient rich growth medium for propagation of Ap, Ac30010, and Ac30234 cells.…”

“…Despite the similarities in co-culture conditions evaluated and the rigorous testing of numerous Francisella and FLA strains in this study, amplification of each Francisella spp. strain in the presence of FLA was not observed (Figures 1-3), which is contrary to the previous studies reporting increases in Francisella CFU densities following co-culture with FLA [33][34][35][36]. Specifically, 3−4 log 10 CFU mL −1 increases of LVS and Fn were observed by days 6−15 in the presence of Ac30010, Ac30234, and Vv at 25−30°C incubation and MOI of 10 [33,35,36].…”

“…These Lp factors have also been shown to be important for Ap, Ac, and Vv infectivity except SidC [59]. In contrast, the LVS and Schu4 mammalian lifecycle entails: (1) entry into host cells via asymmetric spacious pseudopod loops; (2) formation of the Francisella-containing phagosome (FCP); (3) recruitment of early and late endosomal markers to the FCP to avoid lysosome fusion; (4) degradation of the FCP membrane and egress into the host cytosol; and (5) rapid cytosolic growth and depletion of host cell nutrients leading to cellular death and extracellular release of Francisella bacteria (reviewed in [60,61]) which is dependent on the Francisella factors, IglC, MglA, and FTT1103 [62], with IglC and MglA also shown to be important for Ac and Vv intra-amoebae survival [36,63]. These observed differences in the mammalian lifecycles of Lp and Francisella spp., and genetic factors required, could explain the differences in amoeba infectivity between Lp and the Francisella spp.…”

Dissection of Francisella-Host Cell Interactions in Dictyostelium discoideum

“…he genus Francisella comprises Gram-negative, facultative intracellular bacteria that infect a wide range of species, including protozoa, invertebrates, and vertebrates (1)(2)(3). Francisella species can be divided into two lineages represented by Francisella tularensis, causing potentially fatal tularemia in humans, and Francisella noatunensis, infecting aquatic animals (4-6).…”

“…However, these host cell models show major differences in their responses to Francisella infection, such as inflammatory cytokine production (41,43), as well as different interactions with the autophagic pathway (21,25). Therefore, the development of alternative infection systems to study Francisella gained special interest, as indicated by the rising number of models, i.e., Drosophila melanogaster (in vivo and in vitro) (44,45), zebrafish (46,47), and amoeba (1,3). These models may not present all features of the natural host infection, but they complement the currently used systems on the organism and cellular levels.…”